Jun Kazami

Single cell reporter assay using cell surface displayed Vargula luciferase.

Reporter genes such as firefly luciferase are common tools to monitor gene expression in various systems. As reporter gene represents the expression level of the gene of interest with its enzyme activity, firefly luciferase is most frequently used because its luminescent activity is highly sensitive and less time consumable for assay. However, since firefly luciferase is expressed internally in the cell, lysis of the cell is a critical step, and thus it is difficult to monitor the gene expression level continuously. In this report, we utilized secretive Vargula hilgendorfii luciferase modified to cell surface displayed one by fusing with human EGFR transmembrane sequence. This modified Vargula luciferase was expressed on cell surface without losing its bioluminescent activity. Co-transfection with secretive alkaline phosphatase showed that the behaviors of cell surface displayed Vargula luciferase and secretive alkaline phosphatase are comparable to each other. Furthermore, the luminescence of a single cell expressing cell surface displayed Vargula luciferase can be monitored by using photon counting CCD camera, which indicates that this reporter gene can monitor gene expression in a single cell without cell lysis.

Construction and Expression of Chimeric Protein A - Marine Firefly Luciferase in Mammalian Cells

We have constructed novel chimeric proteins that consisted of a single domain of protein A and luciferase originated from marine firefly Vargula hilgendorfii (Umi Hotaru in Japanese) with the aim of obtaining a bifunctional immunological tool. D domain gene of protein A was fused to the 5’ terminus of luciferase cDNA with/without a short linker encoding five amino acids. The resulting constructs were transfected and proteins were expressed in cultured COS-1 or CHO cells either transiently or stably, respectively. The properties of the resultant chimeric protein were characterized by luminometry, immunoblot analysis and affinity binding studies. The results show that the dual biological properties of the chimeric protein could be retained after the introduction of the linker between the two moieties.